The Interaction of Polycationic Organic Polymers with Biological Membranes
NIH RO1 EB005028
Mark M. Banaszak Holl, Ph.D., Principal Investigator, Professor of Chemistry and Macromolecular Science and Engineering, MNIMBS
Ioan Andricioaei, Ph.D., Assistant Professor, Chemistry, University of California at Irvine
James R. Baker Jr., MD, Ruth Dow Doan Professor of Medicine and Bioengineering, Director, MNIMBS
Jolanta Kukowska-Latallo, Ph.D., Research Associate Professor of Internal Medicine, MNIMBS
Anatoli Lopatin, Ph.D., Associate Professor of Molecular and Integrative Physiology
Bradford G. Orr, Ph.D., Professor of Physics, Associate Director, MNIMBS
Thommey P. Thomas, Ph.D., Research Assistant Professor of Internal Medicine, MNIMBS
Nanotechnology holds great promise for biomedical applications such as targeted drug delivery and cell transfection.1-11 The design of optimal materials is hampered by our incomplete understanding of the interaction of nanoparticles with biological membranes. Three major hypotheses have received substantial literature support: 1) energy-dependent endocytosis 2) energy-dependent formation of nanoscale membrane holes 3) energy-independent membrane translocation. It is not clear which mechanistic hypothesis, or combination of hypotheses, best describes the nanoparticle/membrane interactions. In fact, it is possible that the mechanism changes as a function of the cell line and/or physical properties of the nanoparticle under study.
This research program explores the relative significance of these three mechanisms for a promising class of nanoscale drug delivery materials; polycationic organic polymers (POPs). The specific POPs studied include poly(amido)amine (PAMAM) dendrimers, poly-L-lysine (PLL), polyethyleneimine (PEI), and (diethylaminoethyl)-Dextran (DEAE-Dextran).
In addition to assessing the relative importance of the various mechanisms of membrane interaction, the research program will assess the key physical interactions between the POPs and biological membranes that lead to membrane permeability. This issue is particularly important in the context of drug delivery applications since membrane permeability can lead to poor selectivity and/or toxicity. The toxicity implications of the hole forming processes explored in this project are of particular importance and have broad relevance to a wide class of inorganic and organic nanoparticles.
SA1) Assessment of the membrane interaction and transport mechanism of POPs. Experiments designed to assess the relative significance of energy-dependent endocytosis, energy-dependent formation of nanoscale membrane holes, and energy-independent membrane translocation have been performed using a variety of methods including flow cytometry, confocal microscopy, scanning probe microscopy (SPM)/fluoresence microscopy, and patch clamp techniques on living cells in culture.
SA2) Quantification of the relationship between nanoscale hole formation in lipid bilayers and the physical properties of POPs. Systematic variation of the physical properties of PAMAM dendrimers and application of SPM, nuclear magnetic resonance (NMR), and surface plasmon resonace (SPR) to studies on supported lipid bilayers have been employed. These experiments require careful synthesis and analysis of the physical properties of the POPs.
SA3) Determination of the extent and nature of nanoscale hole formation in living cell membranes induced by POPs. Experiments assessing nanoscale holes directly observed in living cells have been carried out.
Studies and Results
The mechanism(s) of POP and polyplex internalization into cells have been explored for two major endocytosis mechanisms. We also examined the relationship of nanoscale membrane holes to internalization, transfection, and expression. The first endocytosis mechanism explored was the GM1/caveolin-1 Lipid Raft Mediated Endocytosis (LRME) mechanism. This mechanism appeared particularly promising since it was reported that the PAMAM dendrimers co-localized with cholera toxin subunit B (CTB), the standard biomarker for LRME. Our initial experiments confirmed the colocalization of CTB and GM1. However, subsequent experiments employing C6 cells, which contain no GM1, demonstrated that internalization was independent of GM1 (Bioconjugate Chemistry 2009, 20, 1503-1513.). We then extended the experiments to polyplexes and once again showed that internalization was GM1 independent. In this case, we also showed that the uptake is also Caveolin-1 independent and that the co-localization with GM1 is an artifact of direct GM1/polyplex interactions (Molecular Pharmaceutics 2010, ASAP).
The initial comparison study of the “Interaction of Polycationic Polymers with Supported Lipid Bilayers and Cells: Nanoscale Hole Formation and Enhanced Membrane Permeability” has been completed and published (Bioconjugate Chemistry 2006, 17, 728-734). This study suggests that nanoscale hole formation in biological membranes is a common property of polycationic polymers, specifically poly-L-lysine (PLL), polyethyleneimine (PEI), diethylaminoethyl-dextran (DEAE-DEX), and poly(amidoamine) (PAMAM) dendrimers.
Using Isothermal Titration Calorimetry (ITC) we have been able to determine the stoichiometry of the dendrimer/lipid interaction and propose a specific structure for the complexes formed (ACS NANO 2009, 3, 1668). Simulations of Dendrimer/Lipid interactions employing the weighted histogram analysis method (WHAM) for determination of the free energy profile and the molecular level interactions that occur between the dendrimer and lipid (J. Phys. Chem. B 2008, 112, 9346; J. Phys. Chem. B 2008, 112, 9337-9345).
Experiments with supported lipid bilayers demonstrated that nanoscale hole formation is a common property of many polycationic nanoscale materials including cationic gold particles, polycationic peptides, and TAT. (Nanoletters 2008, 8, 420). We also were able to directly image PAMAM dendrimers removing lipid via the carpet mechanism (Langmuir 2008, 24, 11003).
Patch clamp studies of PEI, PLL, G7, and G5 PAMAM dendrimers and antimicrobial oligomers with 293A cells provide strong evidence of nanoscale hole formation in living cells. In addition, we have successfully extended the whole cell patch clamp technique to KB cells where the vast majority of our other data has been taken. We have also acquired patch clamp data for polyplexes and also have shown the permeability occurs via the formation of nanoscale holes (J. Phys. Chem. B 2009, 113, 11179).
The research is uncovering fundamental chemical and biological principles need to understand the best approaches to design gene therapy agents. There is also a broader significance for the impact of nanoparticles on human health.
The Mechanism of Polyplexes Internalization into Cells: Testing the GM1/Caveolin-1-Mediated Lipid Raft Mediated Endocytosis Pathway. R. Qi, D. G. Mullen, J. R. Baker, Jr., M. M. Banaszak Holl, accepted Molecular Pharmaceutics.
Cationic Poly(amidoamine) Dendrimer Induces Lysosomal Apoptotic Pathway at Therapeutically Relevant Concentrations. T. P. Thomas, I. Majoros, A. Kotlyar, D. Mullen, M. M. Banaszak Holl, J. R. Baker, Biomacromolecules 2009, 10, 3207-3214.
Cellular Internalization Mechanisms of Poly(amidoamine) Dendrimers: the Role of Ganglioside GM1. S. Hong, R. Rattan, I. J. Majoros, D. G. Mullen, J. L. Peters, X. Shi, A. U. Bielinska, L. Blanco, B. G. Orr, J. R. Baker, M. M. Banaszak Holl. Bioconjugate Chemistry 2009, 20, 1503-1513.
Stoichiometry and structure of poly(amidoamine) dendrimer-lipid complexes. C. V. Kelly, M. G. Liroff, L. D. Triplett, P. R. Leroueil, D.G. Mullen, J. M. Wallace, S. Meshinchi, J. R. Baker, B. G. Orr, M. M. Banaszak Holl. ACS Nano 2009, 3, 1886-1896.
Creation and characterization of blebs on living cells using pulsed laser irradiation. C. V. Kelly, M.-M. T. Kober, P. Kinnunen, D. A. Reis, B. G. Orr, and M. M. Banaszak Holl. J. Bio. Phys. 2009, 35, 279-295.
Polymer Nanoparticles Induce Nanoscale Disruption in Living Cell Plasma Membranes. J. Chen, J. A. Hessler, K. Putchakayala, B. K. Panama, D. P. Khan, S. Hong, D. Mullen, S. C. DiMaggio, A. Lopatin, J. R. Baker, Jr., B. G. Orr, M. M. Banaszak Holl. J. Phys. Chem. B 2009, 113, 11179-11185.
Interactions of Poly(amidoamine) Dendrimers with Survanta Lung Surfactant: The Importance of Lipid Domains. B. Erickson, S. DiMaggio, D. G. Mullen, C. V. Kelly, P. R. Leroueil, S. A. Berry, J. R. Baker, Jr., B. G. Orr, M. M. Banaszak Holl, Langmuir 2008, 24, 11003-11008.
Poly(amidoamine) dendrimers on lipid bilayers II: Effects of bilayer phase and dendrimer termination. C. V. Kelly, P. R. Leroueil, B. G. Orr, M. M. Banaszak Holl, and I. Andricioaei. J. Phys. Chem. B 2008, 112, 9346-9353.
Poly(amidoamine) dendrimers on lipid bilayers I: Free energy and conformation of binding. C. V. Kelly, P. R. Leroueil, E. K. Nett, J. M. Wereszczynski, James R. Baker, B. G. Orr, M. M. Banaszak Holl,and I. Andricioaei. J. Phys. Chem. B 2008, 112, 9337-9345.
Wide Varieties of Cationic Nanoparticles Induce Defects in Supported Lipid Bilayers. P. R. Leroueil, S. A. Berry, K. Duthie, G. Han, V. M. Rotello, D. Q. McNerny, J. R. Baker, Jr.; B. G. Orr, M. M. Banaszak Holl. Nano Letters 2008, 8, 420-424.
Nanoparticle Interaction with Biological Membranes: Does Nanotechnology Present a Janus Face? P. R. Leroueil, S. Hong, A. Mecke, J. R. Baker, Jr., B. G. Orr, M. M. Banaszak Holl Acc. Chem. Res. 2007, 40, 335-342.
Interaction of Polycationic Polymers with Supported Lipid Bilayers and Cells: Nanoscale Hole Formation and Enhanced Membrane Permeability. S. Hong, P. R. Leroueil, E. K. Janus, J. L. Peters, M.-M. Kober, M. T. Islam, B. G. Orr, J. R. Baker, Jr., and M. M. Banaszak Holl. Bioconjugate Chemistry 2006, 17, 728-734.
Physical Interactions of Nanoparticles with Biological Membranes: The Observation of Nanoscale Hole Formation. S. Hong, J. A. Hessler, M. M. Banaszak Holl, P. Leroueil, A. Mecke, B .G. Orr. Chemical Health and Safety 2006, 13, 16-20.
Cell Plasma Membranes and Phase Transitions. M. M. Banaszak Holl in Phase Transitions in Cell Biology ed. by G.H. Pollack and W.-C. Chin, Springer, 2008.
Nanotoxicology: a personal perspective. M. M. Banaszak Holl in Wiley Interdisciplinary Reviews in Nanomedicine, 2009. DOI 10.102/wnan.27
Nanoparticle Interactions with Biological Membranes: A Proposed Physical Mechanism J. R. Baker, B. G. Orr, and M. M. Banaszak Holl in “Nanotoxicology: Characterization, Dosing, and Health Effects” ed by Tran and Monteiro-Riviere, Informa Healthcare USA, New York, 2007.
Nanoparticle-Membrane Interaction: Mechanism for Enhanced Permeability. S. Hong, A. Mecke, P. Leroueil, M. M. Banaszak Holl, and B.G. Orr in “Dendrimer Based Nanomedicine” co-edited by I.J. Majoros and J.R. Baker, World Scientific Publishing, Hackensack, NJ, 2007.